Bottom Line:
We found that majority of amelogenin adsorbed on HAP was released into the surrounding solution by enzymatic processing (88% for MMP20 and 98% for KLK4).The results show that as compared with amelogenin in solution, the HAP-bound amelogenin was hydrolyzed by both MMP20 and KLK4 at significantly higher rates.These results suggest that the adsorption of amelogenin to HAP results in their preferential and selective degradation and removal from HAP by MMP20 and KLK4 in vitro.

Affiliation: Department of Orofacial Sciences, School of Dentistry, University of California, San Francisco San Francisco, CA, USA.

ABSTRACTThe hardest tooth enamel tissue develops from a soft layer of protein-rich matrix, predominated by amelogenin that is secreted by epithelial ameloblasts in the secretory stage of tooth enamel development. During enamel formation, a well-controlled progressive removal of matrix proteins by resident proteases, Matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), will provide space for the apatite crystals to grow. To better understand the role of amelogenin degradation in enamel biomineralization, the present study was conducted to investigate how the adsorption of amelogenin to hydroxyapatite (HAP) crystals affects its degradation by enamel proteinases, MMP20 and KLK4. Equal quantities of amelogenins confirmed by protein assays before digestions, either adsorbed to HAP or in solution, were incubated with MMP20 or KLK4. The digested samples collected at different time points were analyzed by spectrophotometry, SDS-PAGE, high performance liquid chromatography (HPLC), and LC-MALDI MS/MS. We found that majority of amelogenin adsorbed on HAP was released into the surrounding solution by enzymatic processing (88% for MMP20 and 98% for KLK4). The results show that as compared with amelogenin in solution, the HAP-bound amelogenin was hydrolyzed by both MMP20 and KLK4 at significantly higher rates. Using LC-MALDI MS/MS, more accessible cleavage sites and hydrolytic fragments from MMP20/KLK4 digestion were identified for the amelogenin adsorbed on HAP crystals as compared to the amelogenin in solution. These results suggest that the adsorption of amelogenin to HAP results in their preferential and selective degradation and removal from HAP by MMP20 and KLK4 in vitro. Based on these findings, a new degradation model related to enamel crystal growth is proposed.

Figure 1: Removal of bound amelogenin on HAP crystals by MMP-20 and KLK4 hydrolysis. (A) Protein amounts remained on crystals and (B) released into surrounding supernatant at different time points during MMP20 digestion; (C) Protein amounts remained on crystals and (D) released into surrounding supernatant at different time points during KLK4 digestion. H+A, HAP+rh174; H+A+M, HAP+rh174+MMP-20; H+A+K, HAP+rh174+KLK4.

Mentions:
The amount of protein retained on HAP after digestion for various periods of time is illustrated in Figure 1. Both MMP20 and KLK4 hydrolysis could gradually remove the adsorbed amelogenin from HAP (Figures 1A,C) into the reaction buffer solution (Figures 1B,D). After 36 h of digestion by MMP20, only about 12% of amelogenin still remained on HAP. In comparison, the removal rate of adsorbed amelogenin was much faster by KLK4 digestion. Nearly 98% of amelogenin was released into the surrounding solution by merely 12-h processing. During the entire period of incubation with shaking, bound amelogenin could also be desorbed without digestion, but this loss appeared to be negligible compared to that observed in digested samples (Figures 1A,C). Because UV detection showed only a trace amount of MMP20 and KLK4 bound to HAP during digestion process, their effects on the measurement of amelogenin amount were not included from our analysis.

Figure 1: Removal of bound amelogenin on HAP crystals by MMP-20 and KLK4 hydrolysis. (A) Protein amounts remained on crystals and (B) released into surrounding supernatant at different time points during MMP20 digestion; (C) Protein amounts remained on crystals and (D) released into surrounding supernatant at different time points during KLK4 digestion. H+A, HAP+rh174; H+A+M, HAP+rh174+MMP-20; H+A+K, HAP+rh174+KLK4.

Mentions:
The amount of protein retained on HAP after digestion for various periods of time is illustrated in Figure 1. Both MMP20 and KLK4 hydrolysis could gradually remove the adsorbed amelogenin from HAP (Figures 1A,C) into the reaction buffer solution (Figures 1B,D). After 36 h of digestion by MMP20, only about 12% of amelogenin still remained on HAP. In comparison, the removal rate of adsorbed amelogenin was much faster by KLK4 digestion. Nearly 98% of amelogenin was released into the surrounding solution by merely 12-h processing. During the entire period of incubation with shaking, bound amelogenin could also be desorbed without digestion, but this loss appeared to be negligible compared to that observed in digested samples (Figures 1A,C). Because UV detection showed only a trace amount of MMP20 and KLK4 bound to HAP during digestion process, their effects on the measurement of amelogenin amount were not included from our analysis.

Bottom Line:
We found that majority of amelogenin adsorbed on HAP was released into the surrounding solution by enzymatic processing (88% for MMP20 and 98% for KLK4).The results show that as compared with amelogenin in solution, the HAP-bound amelogenin was hydrolyzed by both MMP20 and KLK4 at significantly higher rates.These results suggest that the adsorption of amelogenin to HAP results in their preferential and selective degradation and removal from HAP by MMP20 and KLK4 in vitro.

Affiliation:
Department of Orofacial Sciences, School of Dentistry, University of California, San Francisco San Francisco, CA, USA.

ABSTRACTThe hardest tooth enamel tissue develops from a soft layer of protein-rich matrix, predominated by amelogenin that is secreted by epithelial ameloblasts in the secretory stage of tooth enamel development. During enamel formation, a well-controlled progressive removal of matrix proteins by resident proteases, Matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), will provide space for the apatite crystals to grow. To better understand the role of amelogenin degradation in enamel biomineralization, the present study was conducted to investigate how the adsorption of amelogenin to hydroxyapatite (HAP) crystals affects its degradation by enamel proteinases, MMP20 and KLK4. Equal quantities of amelogenins confirmed by protein assays before digestions, either adsorbed to HAP or in solution, were incubated with MMP20 or KLK4. The digested samples collected at different time points were analyzed by spectrophotometry, SDS-PAGE, high performance liquid chromatography (HPLC), and LC-MALDI MS/MS. We found that majority of amelogenin adsorbed on HAP was released into the surrounding solution by enzymatic processing (88% for MMP20 and 98% for KLK4). The results show that as compared with amelogenin in solution, the HAP-bound amelogenin was hydrolyzed by both MMP20 and KLK4 at significantly higher rates. Using LC-MALDI MS/MS, more accessible cleavage sites and hydrolytic fragments from MMP20/KLK4 digestion were identified for the amelogenin adsorbed on HAP crystals as compared to the amelogenin in solution. These results suggest that the adsorption of amelogenin to HAP results in their preferential and selective degradation and removal from HAP by MMP20 and KLK4 in vitro. Based on these findings, a new degradation model related to enamel crystal growth is proposed.